Fig 2: The effect of ASB6 knockdown or knockout on E-cadherin expression and filopodia formation. The mock-, control shRNA- (shLuc), or ASB6 shRNA- (shASB6#1 and shASB6#2) transduced SAS-M5 cells were analyzed by confocal microscopy immunofluorescence staining of E-cadherin (FITC, green) and nuclei (DAPI, blue) (A). The mock-transfected cells (Mock) and cell pools with different CRISPR/Cas9-directed knockout of ASB6 (ASB6-KO pool#1 and pool#2) were validated by western blots for ASB6 (B), and analyzed by confocal microscopy immunofluorescence staining of F-actin (Phalloidin, red) and nuclei (DAPI, blue). Enlarged images of the regions in the white boxes are shown in the lower panels (C).

Fig 3: The effect of selective media, nutrient-deprivation media, and/or knockdown of ASB6 on the induction of ASB6 and cellular stresses. The SAS cells were grown in selective media (A) or nutrient-deprivation media containing 25% of ingredient of complete media (B) for 0 to 6 or 10 days, and the time-dependent induction of ASB6, ER stress, replication stress, metabolic stress, and autophagy were analyzed by western blots. The control shRNA- (shLuc), or ASB6 shRNA- (shASB6#1 and shASB6#2) transduced SAS-M5 cells were grown in regular or selective media for up to 2 days, and the induction of ASB6 and ER stress were analyzed by western blots. The β-actin and α-tubulin were used as the loading control.

Fig 4: The effect of ASB6 knockdown on the in vivo metastasis. The mock-, control shRNA- (shLuc), or ASB6 shRNA- (shASB6#1 and shASB6#2) transduced SAS-M5 cells were injected into the tail vein of nude mice, and the lung of mice fixed with Bouin's solution were photographed and microscopic examined after H&E staining (magnification 50x) (A), and the pulmonary nodules were quantitated (B). The tumor-free survival of mice was examined by Kaplan-Meier survival analysis (C). * P < 0.05.

Fig 5: The effect of ASB6 overexpression on soft agar colony formation, stemness genes expression, and tumor sphere formation of OSCC cells. The OECM1 cells with stable overexpression of green fluorescent protein (vector GFP) or GFP-tagged ASB6 (ASB6-GFP) were validated by western blot for ASB6 (with as β-actin as the loading control) (A), and were subjected to anchorage-independent growth analysis by the soft agar assay (B), western blots analysis for Nanog and Oct-4 (with as the GAPDH as loading control) (C), and tumor sphere formation analysis (D).* P < 0.05.